The present invention relates to a process for concentrating nucleic acid molecules to be detected of a sample on a surface.
Nucleic acid diagnostics increasingly makes use of “biochips”. They are used, for example, for detecting various types and species of nucleic acids, which may be DNA, RNA, cDNA or other nucleic acids. The biochip-based analytical methods usually aim at detecting special nucleic acids in a sample. Thus, for example, a patient's DNA may be examined for the presence of a particular sequence which indicates a predisposition to a disorder. It is likewise possible to detect pathogens such as viruses and bacteria (e.g. HIV, HPV, HCV) in a patient's blood sample by demonstrating the presence of their DNA or RNA in the sample. These analyses have the advantage of increased accuracy over classical immunoassays, since the pathogen is detected directly rather than via antibodies produced against it.
The biochips used are based in their simplest form on a glass substrate on which capture molecules (e.g. oligonucleotides) are immobilized which can specifically bind to the nucleic acid to be detected. These short nucleic acid fragments which are frequently produced synthetically are, with respect to their sequence, at least partially complementary to the sequence of the nucleic acid to be detected, resulting in highly specific binding. Binding of nucleic acids other than the one to be detected must be avoided in any case in order not to obtain a false-positive result. In many methods, actual detection of the nucleic acid takes place by way of fluorescence processes in which a fluorescent dye is attached to the nucleic acid to be detected, for example via biotinstreptavidin binding. After specific hybridization of the nucleic acid with the capture molecules, the biochip is rinsed to remove unbound material. Consequently, the solution does no longer contain any fluorescent dyes. By exciting the dyes, fluorescence can be observed using a CCD camera, making detection possible.
One advantage of biochips is multiplex detection capability. Thus it is possible to immobilize on various positions of the biochip various kinds of capture molecules targeting various nucleic acids to be detected. Detection can then be carried out by way of space-resolved fluorescence measurement. It is therefore also possible to carry out a plurality of detections of nucleic acids in a single process.
The nucleic acids are copied (amplified) prior to detection, since the number of nucleic acid copies present is usually not sufficient for making direct detection possible. The amplification may be carried out, for example, by a polymerase chain reaction (PCR). PCR is based on replicating nucleic acids with the aid of thermostable DNA polymerases. This involves contacting a pair of oligonucleotide primers (single-stranded oligonucleotides) with the nucleic acid to be amplified. The primers are chosen so as to bind at both ends of a fragment to be amplified on the complementary strands. During elongation the primers are then elongated in the 3′ direction along the particular target nucleic acid strand (forward and reverse primers). Forward and reverse primers are alternatively also referred to as sense or antisense primers. In this way it is possible to amplify the piece located between the sites on the target nucleic acid that are complementary to the primers. For subsequent detection reactions, the PCR products are advantageously removed from primers, nucleotides and other interfering components of the PCR mixture.
The PCR process comprises a plurality of thermocycles, each of which comprises three steps: the sample is first heated (e.g. to 94° C.) in order to separate the strands of the double-stranded target DNA present in the sample (denaturation).
This is followed by lowering the temperature (e.g. to 4560° C.) for the primers to be able to attach to the complementary regions of the now single-stranded DNA (annealing). In the last step, the primers bound to the single strand are extended by DNA polymerase in the 3′ direction according to the information of the DNA template strand, with the corresponding nucleoside triphosphates in the solution being used as building blocks (elongation, e.g. at 72° C.). This cycle is typically run approx. 15-50 times during a PCR. The above temperatures are given merely by way of example and should be adjusted to the PCR to be carried out specifically in each case.
It is thus possible to prepare from a few specimens present of a DNA (or generally of a nucleic acid) a multiplicity of copies within a very short time whose concentration is sufficient for subsequent qualitative detection of the DNA or nucleic acid in the sample. For example, 20 PCR cycles (typically taking 20 to 40 minutes) produce theoretically 220 times, i.e. about 106 times the amount of the nucleic acid originally used. At the same time, PCR enables a label which makes detection possible to be incorporated into the resulting PCR product. Thus it is possible to use, for example, biotin-labeled PCR primers or nucleoside triphosphates, resulting in the synthesized PCR products being biotinylated. After the PCR product has attached to the immobilized capture molecules, biotin is, as a result, likewise immobilized on the biochip at the corresponding position. In a further step streptavidin-linked fluorescent dyes can then be bound to the biotin, which allow the nucleic acid or its PCR product to be detected. Other detection systems, based on electrochemical detection for example, can be used as an alternative to the fluorescent dye.
A central point in biochip-based detection methods is hybridization, i.e. binding of the nucleic acid to be detected (this term is intended herein to be synonymous with the PCR products produced from the nucleic acids). Sensitivity of nucleic acid detection depends on hybridization efficiency. The nucleic acid is usually incubated with the biochip, thereby enabling hybridization to take place. The latter can be improved by choosing a suitable temperature and buffer medium. It is also important that the nucleic acids are moved to the biochip surface occupied with capture molecules for hybridization to be able to take place. It is therefore not optimal, if movement of the nucleic acid is caused merely by thermal diffusion. Thus, for example, the rate constant is only about 8×10−8 cm2/s for a 25mer oligonucleotide (“Observation of hybridization and dehybridization of Thiol-tethered DNA using two-color surface plasmon resonance spectroscopy” Peterlinz et al. (1997), J. Am. Chem. Soc. 119 3401-3402), resulting merely in an average migration of 0.096 cm within a hybridization time of sixteen hours. Thus, assuming that the surface is 4 cm2 in size, passive diffusion alone renders only about 1.5% of the sample nucleic acid accessible to hybridization to the capture molecules immobilized on the biochip. A number of active processes can increase hybridization efficiency.
Thus, C. F. Edman et al. disclose in “Electric field directed nucleic acid hybridization on microchips”, Nucleic Acids Research (1997) 25, 4907-4914, nucleic acids being moved electrophoretically to the biochip by applying an electric field. Hybridization is rendered substantially faster by concentrating the nucleic acid close to the surface of the biochip than by thermal movement of the nucleic acids alone. It is moreover possible to remove non-hybridized nucleic acids from the biochip surface by reversing the electric potential. The method described requires the electrodes to be specially prepared with a protective layer in order to prevent free radicals and changes in pH occurring during the electrode reaction from damaging the concentrated nucleic acids. M. J. Heller et al. disclose a comparable method in “An Active Microelectronics Device for Multiplex DNA Analysis”, IEEE Engineering in Medicine and Biology (1996) March/April, 100-104.
A multiplicity of methods in biochemistry and medical diagnostics now make use of magnetic, polymeric support materials, in particular polymeric particles, for simplifying the removal of cells, proteins and nucleic acids. Compared with conventional separation methods, using magnetic support materials is advantageous in that the loaded support materials can readily and rapidly be removed from the other components of a sample with the aid of magnetic forces. Magnetic bead-shaped or spherical polymeric particles based on polyvinyl alcohol with a narrow particle size distribution within a range of less than 10 μm have proved particularly suitable for such separation methods (WO 9704862).
It is also known that particular biological materials, in particular nucleic acids and proteins, can be isolated from their natural environment only with increased efforts. This is especially due to the fact that mechanical, chemical and biological cell lysis processes must be utilized for isolating the nucleic acids and proteins from the nucleus or the cell membrane or organelles. In addition, the corresponding biological samples usually comprise further, solid and/or dissolved compounds such as other proteins and components of the cytoskeleton, which can impair isolation. An additional difficulty is the fact that very often only small concentrations of the nucleic acids or proteins are present in the biological sample to be studied.
In order to be able nevertheless to utilize the advantages of using magnetic particles for isolating nucleic acids from biological samples, it has been proposed inter alia to isolate nucleic acids with the aid of magnetic particles having a glass surface which is essentially pore-free (WO 9641811). These particles must have a particular composition, i.e. their glass surface must have a particular composition, in order to achieve the desired efficacy. Preparation of these particles moreover requires a relatively complicated process to achieve the necessary sintering of the glass surface.
Known diagnostic processes, for example from nucleic acid and protein diagnostics, usually require a multiplicity of manual operations in order to arrive at an analytical result. This requires inter alia separation of the components to be detected from the rest of the sample. Known examples of separation methods are filtration, centrifugation, chromatography and extraction. All of these are chemical and physical separation processes which are usually not suitable for sequence-specific isolation of DNA or proteins from the sample. Use is made, for example, of resins whose surfaces are functionalized so as to be able to bind DNA or proteins. These target molecules are purified by binding to the solid phase of the resin, followed by a plurality of washing steps and subsequent detachment of the target molecule from the solid phase under suitable buffer conditions. The target molecule must be bound tightly, while contaminating components of the sample are dissolved in a different, liquid phase. After various washing procedures, the target molecule must then be detached again from the solid phase, for example by changing the liquid phase. The repeated change of medium is firstly very costly with regard to material, and secondly product yields fluctuate with each additional process step, making quantitative calibration difficult. In particular in integrated analytical processes, for example lab-on-a-chip systems in which the samples are prepared and analyzed essentially automatically, checking the individual process steps is often not possible, and as a result deviations in the individual process steps amplify each other and may lead to large deviations in the analytical result.
Individual steps can be simplified or even automated completely by the described support particles, also referred to as magnetic beads. The magnetic beads are provided with affinity ligands or other surface modifications and are therefore suitable for binding particular biomolecules, for example DNA, from a solution to their surface. A purification process typically involves adding a suspension of magnetic beads to the sample to be separated in a test tube. After an incubation time of several minutes to enable the affinity ligand to bind to the desired biological molecule, a magnetic field is applied, which removes the particles by accumulating them on one tube wall. The supernatant is discarded and the particles are then washed at least once. For this purpose, the magnetic field is removed first and the particles are suspended in a fresh buffer solution which contains mostly chaotropic salts which prevent the biomolecules from detaching from the support material. The magnetic beads are then deposited on the vessel wall by reapplying the magnetic field. Thus it is possible, after several washing steps, to eluate the molecules by a low salt buffer solution which removes the bound biomolecules from the magnetic beads, in a solution which is free of interfering components, in contrast to the crude extract. The magnetic beads are deposited again on the vessel wall, making available the bio-molecules in the supernatant solution. A disadvantage of the process described is the large amount of liquid required in each case, in the range of several hundred microliters for each individual process step.
Eukaryotic or prokaryotic cells or viruses are known to be isolated, by way of example, by coupling specific antibodies to a fluorescent marker or magnetic beads. The antibody is usually monoclonal and directed to specific binding sites, for example to a surface receptor molecule of a corresponding antigen of the cell or the virus. The desired cells or viruses are labeled by coupling the antibodies to the particular binding site and sorted, for example, by FACS or a permanent magnet. The sorting process can be carried out firstly by way of “positive selection”, involving further processing of the labeled cells or viruses. Secondly, a “negative selection” may be carried out, involving removal of the labeled cells and further processing of the remaining cells. Both methods enable the cells or viruses to be quantified and, as a result, the amounts of reagents required for the further processing to be calculated.
DE 101 11 520 B4 discloses a process for purifying biomolecules with the aid of magnetic particles, which enables in particular relatively small amounts of liquid to be purified in an essentially automated manner. It describes the suspension with magnetic particles being passed through a pipeline which passes by a strong magnetic field. With suitable settings of diameter, flow rate and magnetic field strength, the magnetic particles are deposited on the wall of the pipeline when passing through. The supernatant is discarded by emptying the pipeline or is collected in a receptacle. The arrested particles can then be washed by rinsing with washing solutions. During the washing procedure, the magnetic particles may be held in the pipeline or be suspended and deposited again. The biomolecules are detached from the magnetic particles of the suspension by rinsing with a suitable buffer solution. The pipeline here should be de-signed in such a way that small amounts of liquid of less than 50 μl can also be handled. The described process is particularly suitable for purifying DNA or RNA. The DNA or RNA available in solution at the end of the process may be introduced automatically to a corresponding analytical system. Automation may be carried out by way of a pipetting robot. If the DNA is to be detected via sequence-specific hybridization, it is moreover suggested to run the pipeline additionally over a heating element to achieve denaturation of the DNA double strand. However, in order to analyze DNA using the described process, it is still necessary to extract the DNA from the sample by process steps which have not been described.
Magnetic beads are not only suitable for purifying samples but can also be used for other purposes. Thus, US 2004/0219066 A1 describes a device, by which various particles can be sorted. The particles are bound to different magnetic beads having different magnetic momentums. A magnetic field gradient which moves the magnetic beads, due to their different magnetic momentums, into different collecting boxes is generated in a process chamber. The various particles can thus be distinguished by the differently designed magnetic beads.
WO 00/47983 describes an electrochemical biosensor in which magnetic beads are linked via affinity ligands to components of a sample. An enzyme is coupled to the bound components of the sample and an added substrate is cleaved by the enzyme. The substrate gives rise to a molecule which can be subjected to a redox cycling process. The particular component of the sample can be detected in this way.
Moreover, paramagnetic magnetic beads are known to be used for detecting DNA. Here, capture molecules complementary to the DNA to be detected are located on a magnetorestrictive sensor. If the sample studied contains the DNA to be detected, hybridization between the DNA to be detected and the capture molecules takes place. The hybridized DNA has been or is labeled with biotin to which streptavidin-coated magnetic beads couple. The biotin label is usually introduced into the DNA to be detected by an upstream PCR utilizing bio-tin-labeled primers or nucleotides. After coupling to the paramagnetic beads, the latter are magnetized by an applied magnetic field and their stray field is measured by the magnetoresistive sensor. This results indirectly in quantitative detection of the DNA in the sample.
DE 41 27 657 and WO 9704862, whose disclosure with regard to the methods of preparing support materials is hereby incorporated as reference, disclose processes for preparing magnetic polyvinyl alcohol support materials, preferably in bead-like particle design. According to the disclosed processes, magnetic particles can be prepared with a very narrow particle size distribution and with particle sizes of from 1 to 4 μm, as used in particular for isolating biosubstances in suspension and for diagnostic medicine.
The polyvinyl alcohol particles are prepared by adding particular emulsifier mixtures to the oil phase of the water-in-oil emulsion. Suitable emulsifiers which are added as additives to the oil phase are propylene oxide-ethylene oxide block copolymers, sorbitan fatty esters, mixed complex esters of pentaerythrite fatty esters with citric acid, polyethylene glycol-castor oil derivatives, block copolymers of castor oil derivatives, polyethylene glycols, modified polyesters, polyoxyethylene sorbitan fatty esters, polyoxyethylene-polyoxypropylene-ethylenediamine block copolymers, polyglyceryl derivatives, polyoxyethylene alcohol derivatives, alkylphenyl-polyethylene glycol derivatives, polyhydroxy fatty acid-polyethylene glycol block copolymers, polyethylene glycol ether derivatives. Substances of this kind are commercially known inter alia under the trade name: Pluronic®, Synperonic®, Tetronic®, Triton®, Arlacel®, Span®, Tween®, BrijOR, ReneXOR, Hyperme®, Lameform®, Dehymuls® or Eumulgin®.
To obtain uniform, bead-shaped polymeric particles, preferably with particle sizes of 0.5-10 μm, a mixture of at least two, preferably three to four, of the surfactants is added to the oil phase. Preference is given to mixing a lipophilic emulsifier component with at least one emulsifier which has semihydrophilic properties, i.e. which is soluble in both water and oil. Examples of emulsifiers which meet the latter properties are: ethylene oxide-propylene oxide block copolymer derivatives with a predominant ethylene oxide proportion, polyethylene glycol hexadecyl ethers, shorter-chain polyoxyethylene sorbitan fatty esters, polyethylene glycols or shorter-chain sorbitan fatty esters. The concentration of the emulsifiers in the oil phase is usually 2-6% by volume, preferably 3.5-5.0% by volume. Advantageous with respect to fineness and narrow particle size distribution of polymer droplets are those emulsifier mixtures which comprise at least two lipophilic components and one semihydrophilic emulsifier. The concentration of the semihydrophilic emulsifier is usually between 15 and 30% by volume, based on the total amount of emulsifier. In addition to fineness of the particles, the particles show a bead-like shape.
Apart from the emulsifiers for the oil phase, special surfactants which are soluble in the aqueous polymer phase also contribute to improving the quality of the emulsion, especially of polyvinyl alcohol solutions with low molecular weight (Mowiol, Clariant GmbH, Frankfurt am Main, Del.). In addition, the magnetic colloids added in solid form are successfully finely dispersed by adding ionic emulsifiers. Examples of such emulsifiers which can also be used as binary mixtures are: serum albumin, gelatin, aliphatic and aromatic sulfonic acid derivatives, polyethylene glycols, poly N-vinylpyrrolidone or cellulose acetate butyrate. The amounts of emulsifiers used are usually 0.01-2% by weight, based on the polymer phase, with the concentration of the ionic emulsifiers always being between 0.01 and 0.05% by weight. The skilled worker is familiar with influences of stirrer speeds and of concentrations and viscosities of the two phases on particle size. To obtain the preferred particle sizes of 0.510 μm, stirrer speeds of 1500-2000 revolutions per minute are required, with conventional two blade propeller stirrers being used.
In principle, those ferro- or superparamagnetic colloids which have an appropriate particle size and normally a magnetic saturation of from 50 to 400 Gauss may be used as magnetic particles which are encapsulated into the polyvinyl alcohol matrix during the process. Another requirement to be met by the magnetic particles is dispersibility in the aqueous polymer phase containing the polyvinyl alcohol. During subsequent emulsion in the organic phase, the magnetic colloids are then simultaneously enclosed in the polymer droplets.
Suitable magnetic colloids are preferably magnetites with particle sizes of 10-200 nm. Such substances can be obtained commercially, for example, under the trade name Bayferrox or Ferrofluidics. Since preparing such colloids is general related art, the magnetic particles may also be prepared by the disclosed processes, as described, for example, by Shinkai et al., Biocatalysis, Vol. 5, 1991, 61, or Kondo et al., Appl. Microbiol. Biotechnol., Vol. 41, 1994, 99. The concentrations of the colloids in the polymer phase are, in each case based on this phase, usually between 4 and 14% by volume for colloids which are already aqueous colloids due to their preparation, and 0.3-2% by weight for solid substances. Preparation involves admixing the magnetic colloids directly with the polymer phase. In order to ensure a finely dispersed, even distribution of the particles, brief mixing of the aqueous dispersion by a high revolution dispersing tool (Ultra-Turrax) with subsequent ultrasound treatment is beneficial. The polymer phase required for preparing the magnetic particles usually includes a 2.5-10% by weight polyvinyl alcohol solution.
The magnetic polyvinyl alcohol support material can then be obtained from the suspension by methods known per se to the skilled worker, for example by filtration and washing.
A known process for functionalization comprises equipping the support material with affinity ligands on the surface. This usually requires attaching chemically reactive groups on the surface, to which the affinity ligands are then bound. These groups may be, for example, tosyl, hydroxyl, aldehyde or carboxyl, amino, thiol or epoxy groups. They may generally be provided by treating uncoated monodisperse superparamagnetic particles in order to provide them with a surface layer of a polymer carrying such a functional group, for example a cellulose derivative or a polyurethane together with a polyglycol for providing hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid for providing carboxyl groups or an amino-alkylated polymer for providing amino groups. U.S. Pat. No. 4,654,267 discloses a plurality of surface coatings.
DE 100 13 995 A1 discloses magnetic support materials based on polyvinyl alcohol, whose surface is at least partially silanized and, where appropriate, equipped with biomolecule-coupling affinity ligands. The described support materials may be designed as filter, membrane or particle. Preference is given to the magnetic support material being bead-shaped or spherical particles, the particles having a particle size preferably of from 0.2 to 50 μm, particularly preferably from
0.5 to 5 μm. Aside from the preferably bead-shaped and spherical design of the particles, their particle size distribution should be within as narrow a range as possible. The support materials are preferably prepared in particle form by reacting the polyvinyl alcohol support material with an organic silane compound. The silanized particles are then reacted with affinity ligands.
Affinity ligands which may be coupled are in principle any ligands used in affinity chromatography. Examples of these are: protein A, protein G, protein L, streptavidin, biotin, heparin, antibodies, serum albumin, gelatin, lysine, concanavaline A, oligosaccharides, oligonucleotides, polynucleotides, protein-binding metal ions, lectins, aptamers or enzymes. The special fractionations which can be carried out using such affinity matrices are general related art.
The described advantages of the magnetic beads can also be used for concentrating nucleic acids on biochips, as disclosed by US 2002/0166764 A1. The device described there comprises a biochip which is arranged in a chamber. A liquid flow can be adjusted through the chamber. Outside the chamber, there is a magnetic field generator which enables magnetic beads inside the chamber to be moved to the surface of the biochip. The nucleic acids are specifically linked to the magnetic beads before introduction into the chamber. The linkage is provided by oligonucleotides on the magnetic beads. The magnetic beads, and therefore also the nucleic acids, are then moved in the chamber by the magnetic field generator to the surface of the biochip and held there. The presence of the nucleic acids on the magnetic beads is detected by a redox cycling process. The described process is not capable of multiplexing, since the magnetic beads cannot be addressed to individual positions, as required in microar-rays. The magnetic beads are held unspecifically close to the surface of the biochip.
WO 98/4584 discloses a process for detecting proteins (immunoassays), which likewise makes use of magnetic beads. A capture molecule capable of coupling to a target binding molecule is immobilized on a magnetic bead. The target binding molecule in turn is linked to the protein to be detected. After a complex of the protein, the target binding molecule, the capture molecule and the magnetic bead has formed, it can be separated from the rest of the sample by magnetic forces. Under “mild” conditions which are not specified in any detail, the bond between the capture molecule and the target binding molecule can be dissolved, without the target binding molecule losing its binding ability. The magnetic beads with the capture molecules remaining thereon are removed. The proteins are bound by immobilized antibodies on a surface and are therefore likewise immobilized. Detection molecules, for example fluorescent dyes, are bound to the target binding molecules for detection. Thereby the proteins in the sample can be detected.